3 a - Hydroxysteroid Dehydrogenase Activity of the Y ' Bile Acid Binders in Rat Liver Cytosol

Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3a-hydroxysteroid dehydrogenases (3a-HSD), bile acid-metabolizing enzymes. 3aHSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3a-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3a-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for monoand dihydroxy bile acids were 1-2 ,uM, and cholic acid competitively inhibited the reduction of 3oxo-cholic acid. Under normal redox conditions, partially purified 3a-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3a-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3a-hydroxy bile acids.